Selective amplification of overlapping amplicons

ABSTRACT

The present invention relates to a scalable multiplex PCR method that can simultaneously amplify overlapping amplicons without the drawbacks of conventional multiplex PCR. The method selectively amplifying target nucleic acid fragments having an overlapping region. The method comprises the steps of: obtaining a first nucleic acid sequence comprising a first tag t2 and a first forward primer F1, obtaining a second nucleic acid sequence comprising a second tag t1 and a first reverse primer R1, obtaining a third nucleic acid sequence comprising the second tag t1 and a second forward primer F2, obtaining a fourth nucleic acid sequence comprising a third tag t3 and a second reverse primer R2, wherein each primer is a gene-specific primer; performing initial cycles of PCR; and then performing later cycles of PCR at higher annealing temperatures to obtain amplification products.

This application claims priority to U.S. Provisional Application No.62/129,360, filed Mar. 6, 2015; the content of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

This invention relates to a method for selectively amplifying targetnucleic acid fragments having an overlapping region. The present methodenriches copies of target amplicons over copies of the overlappingregions of amplicons.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of Feb. 23, 2016, anda size of 5.11 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Multiplex-PCR consists of multiple primer sets within a single PCRmixture to produce amplicons that are specific to different DNAsequences. By targeting multiple genes at once, additional informationmay be gained from a single test run that would otherwise requireseveral times of the reagents and more time to perform. Annealingtemperature for each of the primer sets must be optimized to workcorrectly within a single reaction. Commercial kits for multiplexing PCRgeneral reagents are available. The technique of multiplex PCR has beenused for target enrichment for next-generation sequencing (NGS), whichrefers to high throughput parallel DNA sequencing technologies. Millionsor billions of DNA strands can be sequenced concurrently, yieldingsubstantially high throughput.

One major reason for amplicon drop-out is preferential amplification ofthe short overlapping regions between two overlapping amplicons duringamplification. Currently, to amplify two overlapping DNA amplicons, theprimer pairs specifically targeting each amplicon are physicallyseparated into different reaction wells, tubes or micro-droplets. Forexample, BRCA1 and BRCA2 genes contain large exons that require PCRamplification of overlapping DNA amplicons to ensure 100% base coverage.Ion AmpliSeq BRCA1 and BRCA2 Panel, Qiagen GeneRead Human BRCA1 andBRCA2 Panel, and Multiplicom BRCA MASTR Dx separate primer pairs into 3,4, 5 primers pools, respectively, primarily due to the inability toamplify overlapping amplicons efficiently. However, multiple primerpools significantly complicate the workflow and increase the cost oftesting. RainDance Technologies overcomes this issue by separating PCRprimers into thousands of micro-droplets, but on a special expensiveinstrument.

Combining all PCR primers for two overlapping DNA regions in onemultiplex reaction produces four products resulting from four differentcombinations of the two forward primers with the two different reverseprimers. FIG. 1 shows a conventional PCR method. The four PCR products(FIG. 1) are two targeted amplicons (Amplicon 1 and Amplicon 2), onelong amplicon (Amplicon 4_long) spanning the entire region of the twotargeted amplicons and one short amplicon (Amplicon 3_overlap)containing only the overlapped regions between the two targetedamplicons. Using conventional primer design and PCR conditions, duringcycling, the longest amplicon (Amplicon 4_long) serves as DNA templatefor all four amplicons' amplification, and each of the two targetedamplicons (Amplicon 1 & 2) serves as DNA template for amplification ofits own amplicon as well as the shortest amplicon (Amplicon 3_overlap).Assuming that all amplifications occur at 100% efficiency, at PCR cyclen, the amount of the four products—Amplicon 1, Amplicon 2, Amplicon4_long and Amplicon 3_overlap—will be n×2^(n), n×2^(n), 2^(n), andn²×2^(n), respectively. The amount of shortest amplicon (Amplicon3_overlap) is n times higher than that of each of the two targetedamplicons (Amplicon 1 & 2) which in turn is n times higher than theamount of the longest amplicon (Amplicon 4_long).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a traditional multiplex PCR (prior art), foramplification of overlapping target segments.

FIG. 2 illustrates one embodiment of a first round of PCR of the presentinvention, which is the amplification by target-specific primers. F1 andR1 are the forward and reverse primers of target segment 1, while F2 andR2 are the forward and reverse primers of target segment 2. F2^ is apartial sequence of the 5′-end portion of the F2 primer. Tag oligomersof t1, t2 and t3 do not bind to the target. Tags t2 and t3 may share thesame sequences and t1 is different. 1, 2, 3 and 4 indicate theamplification products from the combination of four primers.Amplification of Amplicon 3, the short products from F2 and R1, isinhibited by the formation of a stem-loop structure.

FIG. 3 shows the sizes and locations of Amplicons 1-4 on humanchromosome 13 for Examples 1 and 2.

FIG. 4 shows the agarose gel electrophoresis results of theamplification products after the first round of PCR (Examples 1 and 2).

FIG. 5 is a schematic illustration of the gene-specific amplicons usedin examples 3-6. CDS: protein coding sequence region of a gene; ROI:region of interest. In this example, ROI includes the CDS of BRCA2 geneexon 27 plus 20 bp upstream and 20 bp downstream of xon 27 (CDS±20 bp).

FIG. 6 shows the agarose gel electrophoresis results of amplificationproducts after the first round (Example 3) and second round of PCR(Example 4).

FIG. 7 is a chart showing the coverage data from NGS results ofamplicons (Example 6).

DETAILED DESCRIPTION OF THE INVENTION Definition

An “amplicon” is a piece of DNA or RNA that is the source and/or productof natural or artificial amplification or replication events. In thiscontext, “amplification” refers to the production of one or more copiesof a genetic fragment or target sequence, specifically the amplicon. Asthe product of an amplification reaction, amplicon is usedinterchangeably with common laboratory terms, such as PCR product.

“Locked nucleic acids” (LNA™) are a class of high-affinity RNA analoguesin which the ribose ring is “locked” by a methylene bridge connectingthe 2′-O atom and the 4′-C atom resulting in the ideal conformation forWatson-Crick binding. This modification significantly increases themelting temperature of an oligonucleotide and is also very nucleaseresistant (www.exiqon.com/lna-technology).

“Peptide nucleic acids” (PNAs) are synthetic homologs of nucleic acidsin which the phosphate-sugar polynucleotide backbone is replaced by aflexible pseudo-peptide polymer to which the nucleobases are linked.Because the PNA strand is uncharged, a PNA-DNA duplex will have a highermelting temperature than the corresponding DNA-DNA duplex.

“Universal (PCR) primers” are non-target specific primers that hybridizeto universal tags (non-target specific tags such as e.g. t1, t2 and/ort3 in FIG. 2) flanking both ends of any DNA insert sequences. PCR thatuses universal primers can amplify any DNA inserts that are flanked bytheir complementary tag sequences.

The inventors have discovered a scalable multiplex PCR technology,SLIMAMP™ (Stem-Loop Inhibition-Mediated Amplification), which allows forparallel amplification of hundreds of thousands of amplicons in onetube. This novel multiplex PCR method can simultaneously amplifyoverlapping amplicons without the drawbacks of conventional multiplexPCR, which predominantly amplifies the short overlapping nucleic acidsequences. The present method is a target enrichment method, whichenriches copies of target amplicons over copies of the overlappingregions of amplicons.

The present invention is directed to a method for selectively amplifyingtarget nucleic acid fragments having an overlapping region, without apredominant amplification of the short overlapping nucleic acidsequences. The present invention allows all primers in a single primerpool without introducing any additional expensive equipment.

The present method comprises the steps of: (a) obtaining a first nucleicacid sequence comprising a first tag t2 and a first forward primer F1,(t2F1), complementary to a first target nucleic acid fragment (amplicon1), (b) obtaining a second nucleic acid sequence comprising a second tagt1 and a first reverse primer R1, (t1R1), complementary to the firsttarget nucleic acid fragment (amplicon 1), (c) obtaining a third nucleicacid sequence comprising the second tag t1 and a second forward primerF2, (t1F2), complementary to a second target nucleic acid fragment(amplicon 2), (d) obtaining a fourth nucleic acid sequence comprising athird tag t3 and a second reverse primer R2, (t3R2), complementary tothe second nucleic acid fragment (amplicon 2), wherein the first and thesecond target nucleic acid fragments have an overlapping region, (e)mixing the first and the second target nucleic acid fragments, thefirst, the second, the third, and the fourth nucleic acid sequences, andan effective amount of reagents necessary for performing a polymerasechain reaction (PCR); (f) cycling the mixture of (e) through denaturing,annealing and primer extension steps of PCR for at least two times, and(g) cycling the mixture of (f) through denaturing, annealing and primerextension steps of PCR at an annealing temperature higher than that instep (f) to obtain amplification products. The above method stepsdescribe a first round of PCR cycles.

F1, R1, F2, R2 are gene-specific primers, which are complementary tospecific regions of genomic DNA. The length of these primers can bechosen by a person skilled in the art. In general, the gene-specificprimers are 6-40, 10-50, 10-40, 10-100, 20-40, or 20-50 nucleotides inlength.

Tags t1 and t2 are two different universal tag sequences. Tag t3 canhave the same or different sequence as t2. Tag oligomers of t1, t2 andt3 do not bind to the target sequences. Each tag is at the 5′end of eachgene-specific primer.

The present invention is illustrated in FIG. 2. FIG. 2 is forillustration purposes and is not meant to limit the invention to thedrawings only. The arrangement of tags, forward primers, reverseprimers, target nucleic acids, amplicons as described in steps (a)-(d)is shown at the upper part of FIG. 2. FIG. 2 illustrates one embodimentof the present invention, in which F2^ is a partial sequence of the5′-end portion of the F2 primer. In other embodiments of the invention,F2^ is replaced with 0 nucleotide (not present) or replaced with F2(full sequence of F2 primer) in FIG. 2.

In step (f), the mixture of nucleic acids and reagents go through thePCR cycles of denaturing, annealing and primer extension steps at leasttwo times, such as 2-5 times or 2-10 times, at standard PCR temperaturesor conditions known to a person skilled in the art. In the very firstPCR cycle, amplicons tagged only at one end are generated. In the secondPCR cycle, the one-ended tagged amplicons then serve as templates forthe other tagged primers to generate 2-ended tagged amplicons. Step (f)is illustrated at the middle part of FIG. 2, wherein Amplicon 1 (F1+R1),Amplicon 2 (F2+R2), Amplicon 4_long (F1+R2), and Amplicon 3_overlap(F2+R1) are generated after the initial cycles. After at least twocycles of PCR, complete amplicons with the tag sequences at both endsare generated and ready for the next round of PCR cycles with anincreased annealing temperature of step (g).

In step (g), the mixture of (f) goes through more cycles of PCR ofdenaturing, annealing and primer extension; this time at an annealingtemperature higher than that in step (f), which is an important featureof the present invention. The annealing temperature is increased toprevent the shortest amplicon (Amplicon 3_overlap) to be amplifiedpredominantly.

If the annealing temperature is not increased (as in a conventional PCRmethod), all amplification follows a conventional way where Amplicons 1,2, and 4_long can all serve as templates for the amplification of theshort amplicon (Amplicon 3_overlap); this is because Amplicons 1, 2, and4_long all contain the gene-specific parts of the forward and reverseprimers of Amplicon 3_overlap. As a result of the amplification (withoutincreasing annealing temperature), the amount of the shortest amplicon(Amplicon 3_overlap) is n²×2^(n), which is n times higher than that ofeach of the two targeted amplicons (Amplicons 1 & 2, n×2^(n)), assumingall amplicons have the same amplification efficiencies. In practice, itis observed that amplification efficiency is affected by amplicon lengthin which shorter amplicons correlate with higher amplificationefficiency (Mallona I, et al, BMC Bioinformatics 2011, 12:404), andtherefore, the shorter amplicon amplifies even more favorably.

In step (g) of the present invention, the annealing temperature isincreased, and thus a successful primer-template hybridization requiresnot only the gene-specific parts of the primers, but also the tag parts.When the annealing temperature is increased, Amplicons 1, 2, and 4_longcan no longer serve as templates for the amplification of the shortamplicon (Amplicon 3_overlap), and only the short amplicon itself(Amplicon 3_overlap) can serve as a template for itself. The presentinvention features (i) an increasing annealing temperature after theinitial at least two PCR cycles, and (ii) a proper arrangement of tags(t1, t2, t3) associated with each amplicon, in particular, F2 and R1primers are tagged with the same tag t1; such features result inproduction of 2^(n) copies of all amplicons in theory, because eachamplicon can only use its own amplicon as a template for itsamplification. This is already an improvement over a conventionalmethod, in which Amplicon 3 (short overlap) would yield n times higherthan that of target amplicons. In practice, shorter amplicons typicallyamplify more efficiently than longer amplicons.

In step (g), the annealing temperature is at least 2° C. higher than theannealing temperature in step (f). For example, the annealingtemperature is about 2-35° C., 4-35° C., 5-25° C., 6-20° C., 6-15° C.higher than the gene-specific annealing temperature in step (f). In step(g), the PCR cycling is repeated at least 2 times, e.g., 2-50 times,preferably 2-5, 5-10, 10-30, 10-40, or 10-50 times.

Any added bases can increase the primer's melting temperature when theprimer sequences match the template 100%. In the present invention, thetag sequences are at least 2 or 3 nucleotides in length, and can be5-100, 3-40, 10-30, 10-40, 10-50 nucleotides long. Preferably, tags aredesigned to add at least 5° C. (e.g., 5-10° C. or 5-15° C.) to themelting temperature of the gene-specific untagged primers. The tagsequences provide a higher annealing temperature of the cyclingconditions after the initial minimal 2 cycles in PCR. Tag sequences canbe modified or unmodified nucleic acids. Some modified bases (e.g. LNAor PNA) have higher annealing temperatures than their correspondingnatural bases. When shorter tag sequences are desired for variousreasons, those modified bases can be used instead of the natural bases.

In one embodiment of the invention, the Tm (melting temperature) of tagt1 sequences on both ends of amplicon 3 is high enough to form a tightt1-stem, which prevents the hybridization of primer t1F2 to the amplicon3 template, and inhibits a further exponential amplification of amplicon3. In order to inhibit the binding of primer t1F2 to amplicon 3template, the Tm of the t1-stem at the end of amplicon 3 should be thesame or higher than the Tm of t1F2 oligo alone. The melting andhybridization of t1-stem at the ends of amplicon 3 follow intramolecularkinetics, and are more favorable than those of the regularintermolecular oligo duplex reactions. Therefore, the same two shortcomplimentary oligo sequences (e.g. 2 to 100 nucleotides) have a muchhigher Tm when they form a stem connected by a non-complimentary loop inone molecule than Tm of the same two complimentary oligo sequencesforming a linear duplex. In addition, the Tm of the stem is not onlyinfluenced by the stem sequences but also the loop length (correspondingto the overlapping region). In comparison to a small loop size (e.g.2-200, 5-200, or 10-100 nucleotides), a large loop size (e.g. greaterthan 500 nucleotides such as 500-1000 or 500-1500 nucleotides) reducesthe stem hybridization rates, possibly due to the decreasedprobabilities of contact between the ends of the larger loop, andresembles the kinetics of regular intermolecular DNA duplex formations.

In one embodiment, the second nucleic acid sequence optionally furthercomprises a full sequence (F2), or a partial sequence (F2^)consecutively from the 5′end of the second forward primer (F2), inbetween the second tag (t1) and the first reverse primer (R1). In FIG.2, this optional embodiment of primer t1F2^R1 is shown. Dependent on thelength of F2, in one embodiment, the partial sequence of F2^ is 1-50,1-20, 1-10, or 1-5 nucleotides shorter than F2. For example, the partialsequence of F2^ may be 2-40, 4-40, 8-40, 8-30, or 8-20 nucleotides. Inanother embodiment, the partial sequence of F2^ contains 10-50, 10-90,20-80, 30-70, 40-90, or 50-90% of the F2 sequence.

As shown at the lower part of FIG. 2, after step (g), amplicon 1(F1+R1), Amplicon 2 (F2+R2), and Amplicon 4_long (F1+R2) are amplifiedexponentially by PCR, while the amplification of Amplicon 3_overlap(F2+R1) is inhibited. This is because F2 and R1 primers are tagged withthe same tag t1, and therefore in the presence of F2^ with a properlength, a strong stem loop structure containing the sequences of t1 andF2^ forms and prevents the hybridization of primer t1F2 to the amplicon3 template, which inhibits the further exponential amplification ofamplicon 3. The proper length of F2^ that enables a strong stem loopstructure may vary depending on the Tm of tag t1. FIG. 2 illustrates thestem loop formation with t1 and F2^, which is a preferred embodiment. Inanother embodiment, as described above, the Tm of tag t1 sequences onboth ends of amplicon 3 are high enough to form a tight t1-stem, and thepresence of F2^ is not required.

In another embodiment of the invention, the above amplification productsafter the first round of PCR as described above are amplified further bya second round of PCR amplification with universal primers that bind tot1 and t2 in the first round. In this embodiment, t3 is required to bethe same as t2 during the first round of PCR. The second PCR roundcomprises the steps of: (h) mixing the amplification products from (g),either treated or untreated, with a first and a second universal PCRprimers that bind to t1 and t2 respectively, and an effective amount ofreagents necessary for performing a PCR, wherein both universal PCRprimers do not comprise sequences complementary to the first and thesecond target-specific nucleic acid sequences; and (i) cycling themixture of (h) through denaturing, annealing and primer extension stepsof PCR at standard PCR temperatures and conditions known to a personskilled in the art to obtain second amplification products.

In step (i), the PCR cycling is repeated at least 2 times, e.g., 5-50times, preferably 2-5, 5-10, 10-30, 10-40, or 10-50 times.

During the second round of PCR, the amplifications of the shortoverlapping Amplicon 3_overlap and the long Amplicon 4_long are furtherinhibited because their primer binding sites are blocked by the strongstem formation of t1 at both ends of the Amplicon 3_short and t2 at bothends of the Amplicon 4_long. Therefore, the amplifications of the firstand the second target nucleic acid amplicons (Amplicons 1 and 2)dominate in the second round of PCR. The final products can be used fordifferent purposes, such as next generation sequencing (NGS).

In the second round of PCR, the product of (g) from the first round ofPCR is optionally pre-treated before the step (i), for example, bydilution, single-strand exonuclease digestion, purification, or adaptorligation.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES

Table 1 and FIG. 3 show the sizes and locations of Amplicons 1-4 onhuman genome hg19. Amplicon 3 is the overlap between Amplicons 1 and 2.Amplicon 4 is the long amplicon covering the sequences of both Amplicons1 and 2.

TABLE 1 Size Gene- Size With Chr. Specific Tag Amplicon Name Gene hg19Start hg19 End hg19 (bp) (bp) Amplicon1 BRCA2 chr13 32914076 32914572497 538 Amplicon2 BRCA2 chr13 32914378 32914737 360 401Amplicon3_overlap BRCA2 chr13 32914378 32914572 195 236 Amplicon4_longBRCA2 chr13 32914076 32914737 662 703

Table 2 shows oligonucleotide sequences used in Examples 1 and 2, andFIG. 4 SEQ ID NOs: 1-4 are gene specific primers for BRCA2 amplicon 1and amplicon 2 without tag sequences. SEQ ID NOs: 5-6 are tag sequencesfrom Illumina TSCA tags. SEQ ID NOs: 7-16 are the tagged primers used inthe example experiments.

SEQ ID NO: 7 was used in standard multiplex PCR only (FIG. 4, Lane 3).SEQ ID NOs: 8-14 were used in the present invention (Lane 4-8). SEQ IDNOs: 15-16 were used in both the standard multiplex PCR and the presentinvention (Lane 3-8).

Tm are evaluated by OligoAnalyzer 3.1 (IDTDNA) with 1.5 mM of Mg⁺²concentration.

TABLE 2 Stem_Tm_ formed between Tagged_ t1_F2{circumflex over ( )}_R1Oligo Direction Gene_Specific Full and SEQ ID NO: Sequence* Size_(nt)Amplicon Oligo-Tm OligoTM t1_F2 1: F1 GTGAAAGACATATTTACAGACAGTTTC FWD 27Ampliconl 60.6 N/A N/A 2: R1 CTTGTGAGCTGGTCTGAATGT REV 21 Ampliconl 61.9N/A N/A 3: F2 AGGGAAGCTTCATAAGTCAGTC FWD 22 Amplicon2 61.3 N/A N/A 4: R2TCCTCTAACACTCCCTTAACTTTGT REV 25 Amplicon2 63.1 N/A N/A 5: t1caacgatcgtcgaaattcgc 20 Tag 61.3_TagOnly N/A N/A 6: t2tacacgacgctcttccgatct 21 Tag 64.4_TagOnly N/A N/A 7: t1_F1caacgatcgtcgaaattcgc FWD 47 Amplicon1 60.6 72.6 N/AGTGAAAGACATATTTACAGACAGTTTC 8: t2_F1 tacacgacgctcttccgatct FWD 48Amplicon1 60.6 73.2 N/A GTGAAAGACATATTTACAGACAGTTTC 9: t2_R1tacacgacgctcttccgatct REV 42 Amplicon1 61.9 75.2 N/ACTTGTGAGCTGGTCTGAATGT 10: t1_R1 caacgatcgtcgaaattcgc REV 41 Amplicon161.9 74.7 63.3 (F2{circumflex over ( )} = 0) CTTGTGAGCTGGTCTGAATGT11: t1_F2^4_ caacgatcgtcgaaattcgc AGGG REV 45 Amplicon1 61.9 76.5 75.2R1 CTTGTGAGCTGGTCTGAATGT 12: t1_F2{circumflex over ( )}8_caacgatcgtcgaaattcgc AGGGAAGC REV 49 Amplicon1 61.9 77.2 77.2 R1CTTGTGAGCTGGTCTGAATGT 13: t1_F2{circumflex over ( )}12_caacgatcgtcgaaattcgc AGGGAAGCTTCA REV 53 Amplicon1 61.9 77.2 77.8 R1CTTGTGAGCTGGTCTGAATGT 14: t1_F2{circumflex over ( )}16_  caacgatcgtcgaaattcgc REV 57 Amplicon1 61.9 77.3 77.5 R1 AGGGAAGCTTCATAAGCTTGTGAGCTGGTCTGAATGT 15: t1_F2 caacgatcgtcgaaattcgc FWD 42 Amplicon261.3 73.4 N/A AGGGAAGCTTCATAAGTCAGTC 16: t2_R2 tacacgacgctcttccgatctREV 46 Amplicon2 63.1 74.5 N/A TCCTCTAACACTCCCTTAACTTTGT *Lower caseindicates tag sequences; Underline indicates inserted partial F2sequences in R1; un-labeled upper case sequences are gene-specificsequences. N/A: Not applicable. FWD: forward. REV: reverse

Table 3 shows primer mix information in Examples 1 and 2.

TABLE 3 F2_R1 Short Amplicon Multiplex Primer Mix Loop Length_(nt); StemLane Name Amplicon1 Amplicon2 Oligo; Stem length_(nt) 1 M: 100 bp Ladder2 Singleplex Control Not applicable mix* 3 STD_Multiplex_ctrl t1_F1t2_R1 t1_F2 t2_R2 no stem loop 4 Stem_t1-F2∧0 t2_F1 t1_R1 t1_F2 t2_R2195; t1_only; 20 5 Stem_t1-F2∧4 t2_F1 t1_F2∧4_R1 t1_F2 t2_R2 195; t1 + 4nt of F2; 24 6 Stem_t1-F2∧8 t2_F1 t1_F2∧8_R1 t1_F2 t2_R2 195; t1 + 8 ntof F2; 28 7 Stem_t1-F2∧12 t2_F1 t1_F2∧12_R1 t1_F2 t2_R2 195; t1 + 12 ntof F2; 32 8 Stem_t1-F2∧16 t2_F1 t1_F2∧16_R1 t1_F2 t2_R2 195; t1 + 16 ntof F2; 36 *Contains each singleplex product from separate amplificationsmixed at the equal volume ratio

Example 1: First Round of PCR Amplification (Gene-Specific Primers)

A representative PCR mixture of 25 μL included the following components:12.5 μL of 2× Multiplex Master Mix (KAPA Biosystems, Cat#KK5802), 2 μLhuman genomic DNA (Promega, Cat#G3041) diluted to 5 ng/μL in low TEbuffer (USB, Cat#75793), 6.5 μL nuclease-free water, and 4 μL ofgene-specific primer mix (1.25 μEM each, see Multiplex Primer Mix Lanes3-8 in Table 3).

The singleplex (FIG. 4, Lane 2) PCR, the standard multiplex (FIG. 4,Lane 3) PCR, and the stem-forming multiplex (FIG. 4, Lanes 4-8) PCR wereall performed on a thermal cycler as follows:

1 cycle 95° C.  2 minutes Enzyme activation and initial DNA denaturation 5 cycles 95° C. 30 seconds Denaturation 60° C. 90 secondsAnnealing/extension 30 cycles 95° C. 30 seconds Denaturation 72° C. 90seconds Annealing/extension at an increased temperature 1 cycle 72° C. 5 minutes Final extension 1 cycle  8° C. Hold

Example 2: Agarose Gel Electrophoresis

The products from example 1 were analyzed on an E-base device (LifeTechnologies). Two μL of the product was diluted to a final volume of 20μL with nuclease-free water and loaded onto a 2% SizeSelect E-gel. DNAelectrophoresis of diluted PCR products (Lanes 2-8) and 1 Kb Plus DNAladder (Invitrogen, Cat#10488-090, Lane 1) was performed, and at the endof the run, a digital image of the gel was captured by an E-gel Imager(Life Technologies). Results are shown in FIG. 4.

In Lane 2 of FIG. 4, equal amounts of the products of singleplexreactions from Example 1 are mixed and can be seen together on the gel,designated Amplicons 1 and 2. Standard multiplex PCR predominantlyproduces Amplicon 3 (Lane 3), which is PCR product amplified from theoverlap of Amplicons 1 and 2. Amplicon 4, the entire region covered byAmplicons 1 and 2, can faintly be seen in Lane 3. Lanes 4 and 5, whichcontain stem oligonucleotides of t1 only and t1 plus partial F2 (4nucleotides from the 5′ end), respectively, show similar patterns withthree detectable bands: Amplicons 1, 2 and 3. In Lanes 6-8, allAmplicons 1, 2, and 3 can be seen, but amplicon 3 has decreasedsubstantially relative to Lanes 3-5.

Table 4 and FIG. 5 show the amplicon sizes and locations on human genomehg19 targeting BRCA2 Exon 27 (B2X27) used in examples 3-6.

TABLE 4 Amplicon Amplicon Size Size Gene- With Amplicon Specific TagName chr_hg19 Start hg19 End hg19 (bp) (bp) B2X27A1 chr13 3297218932972455 267 308 B2X27A2 chr13 32972396 32972719 324 365 B2X27A3 chr1332972638 32972985 348 389 Overlap of chr13 32972396 32972455 60 101B2X27A1 + A2 Overlap of chr13 32972638 32972719 82 123 B2X27A2 + A3

Table 5 shows oligonucleotide sequences used in Examples 3, 4, 5, and 6for BRCA2 (B2) Exon 27 (X27) amplification.

TABLE 5 Oligo Direction SEQ ID NO: Sequence* Size (nt) Amplicon 17:caacgatcgtcgaaattcgc ACTGTGTGTAATATTTGCGTGCTT FWD 44 B2X27A1 t1_B2X27A1F18: tacacgacgctcttccgatct TTCTCTTTTTGCAGTTCTTTTGGT REV 45 B2X27A1t2_B2X27A1R 19: caacgatcgtcgaaattcgc CTTCAAAGTCTTGTAAAGGGGAGA FWD 44B2X27A2 t1_B2X27A2F 20: tacacgacgctcttccgatct TTGAACCAGACAAAAGAGCTTGREV 43 B2X27A2 t2_B2X27A2R 21:caacgatcgtcgaaattcgc GAAATTTCTCTTTTGGAAAGTAATTCA FWD 47 B2X27A3t1_B2X27A3F 22: tacacgacgctcttccgatct TGTGGTTTGAAATTATATTCCAGTCTT REV 48B2X27A3 t2_B2X27A3R 23: t2_B2X27A1. tacacgacgctcttccgatct CTTCAAAGTCREV 55 B2X27A1 SLIM.R TTCTCTTTTTGCAGTTCTTTTGGT 24: t2_B2X27A2.tacacgacgctcttccgatct CTTCAAAGTCTTGTAAAGGGGAGA FWD 45 B2X27A2 SLIM.F25: t1_B2X27A2. caacgatcgtcgaaattcgc GAAATTTCTCT REV 53 B2X27A2 SLIM.RTTGAACCAGACAAAAGAGCTTG *Lower case indicates tag sequences; Underlineindicates inserted partial forward primer sequences from the nextamplicon; un-labeled, upper case sequences are gene-specific sequences.

Example 3: First Round of Gene-specific PCR Amplification for BRCA2 exon27 (B2X27)

A representative PCR mixture of 25 μL included the following components:12.5 μL of 2× Multiplex Master Mix (KAPA Biosystems, Cat#KK5802), 6 uLof DNA (Coriell, Cat#NA19240 or NA14622) diluted to 5 ng/μL in low TEbuffer (IDT, Cat#11-05-01-09), 2.5 μL nuclease-free water, and 4 μL ofgene-specific primer mix (1.25 μM each).

The conventional primer mix contained the following six oligos fromTable 5: SEQ ID NOs: 17 and 18 (B2X27 amplicon 1), 19 and 20 (B2X27amplicon 2), and 21 and 22 (B2X27 amplicon 3). The SLIMAMP™ primer mix(the present invention) contains the following six oligos: 17 and 23(B2X27 amplicon 1), 24 and 25 (B2X27 amplicon 2), and 21 and 22 (B2X27amplicon 3).

The standard multiplex and stem-forming multiplex PCR is performed on athermal cycler as follows:

1 cycle 95° C. 2 minutes Enzyme activation and initial DNA denaturation 5 cycles 95° C. 15 seconds  Denaturation 60° C. 6 minutesAnnealing/extension 25 cycles 95° C. 30 seconds  Denaturation 72° C. 3minutes Annealing/extension at increased temperature 1 cycle  8° C. Hold

Example 4: Second Round of PCR Amplification (Universal Primers) andPurification

The gene-specific products in Example 3 were diluted 1000-fold. Arepresentative PCR mixture of 25 μL included the following components:2.5 μL 10× reaction buffer, 0.5 μL dNTPs, 0.25 μL enzyme (Roche,Cat#12140314001), 2 μL of the diluted product from Example 3, 2 μL ofIllumina Index Forward primer, 2 μL Illumina Index Reverse primer (25 μMprimer stock from TruSeq Custom Amplicon Index Kit, Cat#FC-130-1003),and 15.75 μL nuclease-free water.

PCR amplification was performed as follows:

1 cycle 95° C.  4 minutes Initial DNA denaturation 95° C. 30 secondsDenaturation 20 cycles 66° C. 30 seconds Annealing 72° C. 60 secondsExtension 1 cycle 72° C.  5 minutes Final extension 1 cycle  8° C. Hold

The product was purified by adding 18 μL of Agencourt AMPure XP beads(Beckman Coulter, Cat#A63881), separating the beads from thesupernatant, and discarding the supernatant. Two washes of 70% ethanolwere used to wash the beads, and the product was eluted from the beadsusing 32 μL nuclease-free water. The concentration of the product wasthen quantified using 2 μL of the product diluted in 198 μL of QubitHigh Sensitivity buffer (Invitrogen, Cat#Q32854).

Example 5: Agarose Gel Electrophoresis

The products from example 3 and example 4 were analyzed on an E-basedevice (Life Technologies). Two μL of the product was diluted to a finalvolume of 20 μL with nuclease-free water and loaded onto a 2% SizeSelectE-gel. DNA electrophoresis of diluted PCR products and 50 bp DNA ladder(Invitrogen, Cat#10488-043) was performed. At the end of the run, adigital image of the gel was captured by an E-gel Image (LifeTechnologies). Results are shown in FIG. 6.

The products from example 3 (First round of PCR products) are shown inLane 1-4 of FIG. 6. Lanes 1 and 2 demonstrate the products ofconventional multiplex PCR. The small amplicons, which are the overlapsof amplicons 1 with 2 and 2 with 3, were produced along with amplicons1-3. On the other hand, SLIMAMP™ reactions, loaded in Lanes 3 and 4,produce mainly amplicons 1-3, with very little of the overlappingamplicons.

The products from Example 4 (second round of PCR products) are shown inLanes 5-8 of FIG. 6, which are products after universal PCR and cleanedup by beads. In Example 4 (Lanes 5-8), amplicons were tagged andamplified further, demonstrated by the increase in nucleotide size whencompared with Lanes 1-4. The samples that originally underwentconventional multiplex PCR produced primarily the undesired shortoverlapping amplicons (Lanes 5-6), while the SLIMAMP™ samples (thepresent invention) contain only the targeted amplicons 1-3 (Lanes 7-8).

Example 6: NGS Library Normalization and Sequencing

Each product from Example 4 was normalized to 4 mM using 10 mM Tris-HClw/0.1% Tween 20 (Teknova, Cat#T7724). All normalized products fromExample 4 are mixed in equal volume (3 μL each) to create a library mix.5 μL of the library mix is added to 5 μL of 0.2 N NaOH to denature thelibrary. A 20 pM library is prepared using HT1 buffer, loaded, andsequenced with a 250 bp paired-end read length (Illumina,Cat#MS-102-2003). The resulting Fastq sequencing reads for each samplewas then aligned to the hg19 reference genome by BWA-MEM. The paired-endreads were then merged and the coverages for each amplicon regions wereanalyzed. The coverage information is shown in FIG. 7.

FIG. 7 depicts the percentage of NGS reads attributed to each ampliconin each sample. In the conventional multiplex PCR, the overlappingamplicons were amplified more efficiently than the target amplicons andcount for 78% of the total NGS reads, due to their much smaller sizes(60 and 82 bp). In the SLIMAMP™ samples, all the undesired overlappingamplicons were inhibited with close to 0% detected in NGS. With respectto the targeted amplicons in both the conventional and SLIMAMP™ samples,as expected, amplicon 1 (267 bp) had higher percentage than bothamplicons 2 (324 bp) and 3 (348 bp) because a smaller size (amplicon 1)typically amplifies more efficiently.

It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims.

What is claimed is:
 1. A method for selectively amplifying targetnucleic acid fragments having an overlapping region, comprising thesteps of: (a) obtaining a first nucleic acid sequence comprising a firsttag (t2) and a first forward primer (F1) complementary to a first targetnucleic acid fragment, (b) obtaining a second nucleic acid sequencecomprising a second tag (t1) and a first reverse primer (R1)complementary to the first target nucleic acid fragment, (c) obtaining athird nucleic acid sequence comprising the second tag (t1) and a secondforward primer (F2) complementary to a second target nucleic acidfragment, (d) obtaining a fourth nucleic acid sequence comprising athird tag (t3), and a second reverse primer (R2) complementary to thesecond nucleic acid fragment, wherein the first and the second targetnucleic acid fragments have an overlapping region, (e) mixing the firstand the second target nucleic acid fragments, the first, the second, thethird, and the fourth nucleic acid sequences, and an effective amount ofreagents necessary for performing a polymerase chain reaction (PCR); (f)cycling the mixture of (e) through denaturing, annealing and primerextension steps of PCR for at least two times, and (g) cycling themixture of (f) through denaturing, annealing and primer extension stepsof PCR at an annealing temperature higher than that in step (f) toobtain amplification products.
 2. The method according to claim 1,wherein the second nucleic acid sequence further comprises a fullsequence of the second forward primer (F2), in between the second tag(t1) and the first reverse primer (R1).
 3. The method according to claim1, wherein the second nucleic acid sequence further comprises F2^, whichis a 5′-end partial sequence of the second forward primer (F2), inbetween the second tag (t1) and the first reverse primer (R1).
 4. Themethod according to claim 3, wherein the 5′-end partial sequence of thesecond forward primer contains 10-90% of the F2 sequence.
 5. The methodaccording to claim 1, wherein the annealing temperature in step (g) is2-35° C. higher than the annealing temperature in step (f).
 6. Themethod according to claim 1, wherein the PCR cycling in step (f) isrepeated 2-10 times.
 7. The method according to claim 1, wherein thethird tag is the same as the second tag.
 8. The method according toclaim 6, further comprising the steps of: (h) mixing the amplificationproducts from step (g), either treated or untreated, with a first and asecond universal PCR primers that bind to t1 and t2 respectively, but donot bind to the first and the second target nucleic acid fragments, andan effective amount of reagents necessary for performing a PCR, and (i)cycling the mixture of step (h) through denaturing, annealing and primerextension steps of PCR to obtain second amplification products.
 9. Themethod of claim 8, wherein the products from step (g) are pre-treated bydilution, single-strand exonuclease digestion, purification, or adaptorligation.
 10. The method of claim 3, wherein F2^ contains 2-40nucleotides.
 11. The method of claim 3, wherein F2^ contains 4-40nucleotides.
 12. The method of claim 3, wherein F2^ is 1-5 nucleotidesshorter than F2.